Formulations of (r)-1-(2,2-difluorobenzo[d] [1,3] dioxol-5-yl)-n-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1h-indol-5-yl)cyclopropanecarboxamide

ABSTRACT

The present invention relates to formulations of (R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide (Compound 1), pharmaceutical packs or kits thereof, and methods of treatment therewith.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. provisional patent applicationSer. Nos. 61/352,512, filed Jun. 8, 2010, the entire contents of whichis incorporated herein by reference.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to an oral formulation comprising(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(Compound 1) as described herein, water, and a polyethylene glycol(PEG). The oral formulation may additionally comprise a surfactant, andadditionally a taste masker. The invention further relates to a methodof treating a CFTR mediated disease such as cystic fibrosis with such aformulation.

BACKGROUND OF THE INVENTION

CFTR is a cAMP/ATP-mediated anion channel that is expressed in a varietyof cells types, including absorptive and secretory epithelia cells,where it regulates anion flux across the membrane, as well as theactivity of other ion channels and proteins. In epithelia cells, normalfunctioning of CFTR is critical for the maintenance of electrolytetransport throughout the body, including respiratory and digestivetissue. CFTR is composed of approximately 1480 amino acids that encode aprotein made up of a tandem repeat of transmembrane domains, eachcontaining six transmembrane helices and a nucleotide binding domain.The two transmembrane domains are linked by a large, polar, regulatory(R)-domain with multiple phosphorylation sites that regulate channelactivity and cellular trafficking.

The gene encoding CFTR has been identified and sequenced (See Gregory,R. J. et al. (1990) Nature 347:382-386; Rich, D. P. et al. (1990) Nature347:358-362), (Riordan, J. R. et al. (1989) Science 245:1066-1073). Adefect in this gene causes mutations in CFTR resulting in cysticfibrosis (“CF”), the most common fatal genetic disease in humans. Cysticfibrosis affects approximately one in every 2,500 infants in the UnitedStates. Within the general United States population, up to 10 millionpeople carry a single copy of the defective gene without apparent illeffects. In contrast, individuals with two copies of the CF associatedgene suffer from the debilitating and fatal effects of CF, includingchronic lung disease.

In patients with cystic fibrosis, mutations in CFTR endogenouslyexpressed in respiratory epithelia lead to reduced apical anionsecretion causing an imbalance in ion and fluid transport. The resultingdecrease in anion transport contributes to enhance mucus accumulation inthe lung and the accompanying microbial infections that ultimately causedeath in CF patients. In addition to respiratory disease, CF patientstypically suffer from gastrointestinal problems and pancreaticinsufficiency that, if left untreated, results in death. In addition,the majority of males with cystic fibrosis are infertile and fertilityis decreased among females with cystic fibrosis. In contrast to thesevere effects of two copies of the CF associated gene, individuals witha single copy of the CF associated gene exhibit increased resistance tocholera and to dehydration resulting from diarrhea—perhaps explainingthe relatively high frequency of the CF gene within the population.

Sequence analysis of the CFTR gene of CF chromosomes has revealed avariety of disease-causing mutations (Cutting, G. R. et al. (1990)Nature 346:366-369; Dean, M. et al. (1990) Cell 61:863:870; and Kerem,B-S. et al. (1989) Science 245:1073-1080; Kerem, B-S et al. (1990) Proc.Natl. Acad. Sci. USA 87:8447-8451). To date, greater than 1000disease-causing mutations in the CF gene have been identified asreported by the scientific and medical literature. The most prevalentmutation is a deletion of phenylalanine at position 508 of the CFTRamino acid sequence, and is commonly referred to as ΔF508-CFTR. Thismutation occurs in approximately 70 percent of the cases of cysticfibrosis and is associated with a severe disease. Other mutationsinclude the R117H and G551D.

The deletion of residue 508 in ΔF508-CFTR prevents the nascent proteinfrom folding correctly. This results in the inability of the mutantprotein to exit the ER, and traffic to the plasma membrane. As a result,the number of channels present in the membrane is far less than observedin cells expressing wild-type CFTR. In addition to impaired trafficking,the mutation results in defective channel gating. Together, the reducednumber of channels in the membrane and the defective gating lead toreduced anion transport across epithelia leading to defective ion andfluid transport. (Quinton, P. M. (1990), FASEB J. 4: 2709-2727). Studieshave shown, however, that the reduced numbers of ΔF508-CFTR in themembrane are functional, albeit less than wild-type CFTR. (Dalemans etal. (1991), Nature Lond. 354: 526-528; Denning et al., supra; Pasyk andFoskett (1995), J. Cell. Biochem. 270: 12347-50). In addition toΔF508-CFTR, other disease causing mutations in CFTR that result indefective trafficking, synthesis, and/or channel gating could be up- ordown-regulated to alter anion secretion and modify disease progressionand/or severity.

Although CFTR transports a variety of molecules in addition to anions,it is clear that this role (the transport of anions) represents oneelement in an important mechanism of transporting ions and water acrossthe epithelium. The other elements include the epithelial Na⁺ channel,ENaC, Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and the basolateralmembrane K⁺ channels, that are responsible for the uptake of chlorideinto the cell.

These elements work together to achieve directional transport across theepithelium via their selective expression and localization within thecell. Chloride absorption takes place by the coordinated activity ofENaC and CFTR present on the apical membrane and the Na⁺—K⁺-ATPase pumpand Cl— channels expressed on the basolateral surface of the cell.Secondary active transport of chloride from the luminal side leads tothe accumulation of intracellular chloride, which can then passivelyleave the cell via Cl⁻ channels, resulting in a vectorial transport.Arrangement of Na⁺/2Cl⁻/K⁺ co-transporter, Na⁺—K⁺-ATPase pump and thebasolateral membrane K⁺ channels on the basolateral surface and CFTR onthe luminal side coordinate the secretion of chloride via CFTR on theluminal side. Because water is probably never actively transporteditself, its flow across epithelia depends on tiny transepithelialosmotic gradients generated by the bulk flow of sodium and chloride.

As discussed above, it is believed that the deletion of residue 508 inΔF508-CFTR prevents the nascent protein from folding correctly,resulting in the inability of this mutant protein to exit the ER, andtraffic to the plasma membrane. As a result, insufficient amounts of themature protein are present at the plasma membrane and chloride transportwithin epithelial tissues is significantly reduced. In fact, thiscellular phenomenon of defective endoplasmic reticulum (ER) processingof ATP-binding cassette (ABC) transporters by the ER machinery, has beenshown to be the underlying basis not only for CF disease, but for a widerange of other isolated and inherited diseases. The two ways that the ERmachinery can malfunction is either by loss of coupling to ER export ofthe proteins leading to degradation, or by the ER accumulation of thesedefective/misfolded proteins [Aridor M, et al., Nature Med., 5(7), pp745-751 (1999); Shastry, B. S., et al., Neurochem. International, 43, pp1-7 (2003); Rutishauser, J., et al., Swiss Med Wkly, 132, pp 211-222(2002); Morello, J P et al., TIPS, 21, pp. 466-469 (2000); Bross P., etal., Human Mut., 14, pp. 186-198 (1999)].

(R)-1-(2,2-Difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamideis disclosed in International PCT Publication WO 2010054138 (saidpublication being incorporated herein by reference in its entirety) as amodulator of CFTR activity and thus as a useful treatment forCFTR-mediated diseases such as cystic fibrosis. A need remains, however,for pharmaceutical compositions comprising(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidethat are readily prepared and that are suitable for use as therapeutics.

SUMMARY OF THE INVENTION

The present invention relates to oral formulations of(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamidewhich has the structure below:

Compound 1 is useful for treating or lessening the severity of a varietyof CFTR mediated diseases.

In one aspect, the invention features a formulation comprising(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(Compound 1), water, and a polyethylene glycol (PEG).

In another embodiment, the polyethylene glycol is selected from PEG 300,PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, or PEG 1000. Inanother embodiment, the polyethylene glycol is PEG 400.

In another embodiment, the concentration of Compound 1 is from about0.05 to about 3% by weight. In another embodiment, the concentration ofCompound 1 is from about 0.1 to about 2% by weight. In anotherembodiment, the concentration of Compound 1 is about 0.12, 0.25, or0.50% by weight.

In another embodiment, the concentration of polyethylene glycol is fromabout 40 to about 60% by weight. In another embodiment, theconcentration of polyethylene glycol is from about 45 to about 55% byweight. In another embodiment, the concentration of polyethylene glycolis about 50% by weight.

In another embodiment, the concentration of Compound 1 is from about0.05 to about 3% by weight; and the concentration of polyethylene glycolis from about 40 to about 60% by weight. In another embodiment, theconcentration of Compound 1 is from about 0.1 to about 2% by weight; andthe concentration of polyethylene glycol is from about 45 to about 55%by weight. In another embodiment, the concentration of Compound 1 isabout 0.12, 0.25, or 0.50% by weight; and the concentration of ethyleneglycol is about 50% by weight.

In another embodiment, the concentration of Compound 1 is about 0.12,0.25, or 0.50% by weight; and the polyethylene glycol is PEG 400 atabout 50% by weight.

In another embodiment the formulation of Compound 1 further comprises asurfactant. In another embodiment, the surfactant is an anionic,cationic, or nonionic surfactant. In another embodiment, the surfactantis a nonionic surfactant selected from the group consisting of vitamin Ed-α-tocopheryl PEG 1000 succinate (vitamin E TPGS), polysorbate 20,polysorbate 40, polysorbate 60, polysorbate 65, polysorbate 80, alkylpoly(ethylene oxide), poloxamine, alkyl polyglucosides, octyl glucoside,decyl maltoside, fatty alcohol, cetyl alcohol, oleyl alcohol, cocamideMEA, cocamide DEA, Solutol surfactants such as Solutol HS 15, andcocamide TEA. In another embodiment, the surfactant is vitamin E TPGS.

In another embodiment, the concentration of surfactant is from about 5to about 15% by weight. In another embodiment, the concentration ofsurfactant is from about 10 to about 12% by weight. In anotherembodiment, the concentration of surfactant is about 11% by weight. Inanother embodiment, the surfactant is vitamin E TPGS at about 11% byweight.

In another embodiment, the concentration of Compound 1 is from about0.05 to about 3% by weight, the concentration of polyethylene glycol isfrom about 40 to about 60% by weight, and the concentration ofsurfactant is from about 5 to about 15% by weight.

In another embodiment, the concentration of Compound 1 is from about0.05 to about 3% by weight, the polyethylene glycol is PEG 400 at about40 to about 60% by weight, and the surfactant is vitamin E TPGS at about5 to 15% by weight.

In another embodiment the formulation of Compound 1 is about 30 to 50 g.In another embodiment, the formulation is about 35 to 45 g. In anotherembodiment, the formulation is about 40 g.

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (g) Compound 1 0.05 PEG 400 19.95 Vitamin E TPGS 4.48Water 15.52

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (g) Compound 1 0.10 PEG 400 19.90 Vitamin E TPGS 4.48Water 15.52

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (g) Compound 1 0.20 PEG 400 19.80 Vitamin E TPGS 4.48Water 15.52

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (g) Compound 1 0.30 PEG 400 19.70 Vitamin E TPGS 4.48Water 15.52

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (% by weight) Compound 1 0.10-0.80 PEG 400 48-51Vitamin E TPGS 10-12 Water 37-40

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (% by weight) Compound 1 0.20-0.60 PEG 400 49-50Vitamin E TPGS 10-12 Water 37-40

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (% by weight) Compound 1 0.25 PEG 400 49.75 Vitamin ETPGS 11.20 Water 38.80

In another embodiment, the formulation of Compound 1 comprises thefollowing:

Amount Component (% by weight) Compound 1 0.50 PEG 400 49.50 Vitamin ETPGS 11.20 Water 38.80

In another embodiment, the invention features any of the above describedformulations further comprising a taste masker. In another embodiment,the invention features any of the above described formulations furthercomprising an additional therapeutic agent. In another embodiment, theadditional therapeutic agent is selected from a mucolytic agent,bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than Compound 1, or anutritional agent.

In another aspect, the invention features a method of treating a CFTRmediated disease selected from cystic fibrosis, asthma, smoke inducedCOPD, chronic bronchitis, rhinosinusitis, constipation, pancreatitis,pancreatic insufficiency, male infertility caused by congenitalbilateral absence of the vas deferens (CBAVD), mild pulmonary disease,idiopathic pancreatitis, allergic bronchopulmonary aspergillosis (ABPA),liver disease, hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, protein C deficiency, Type 1hereditary angioedema, lipid processing deficiencies, familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, 1-cell disease/pseudo-Hurler,mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetesinsipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders, Huntington's, spinocerebullar ataxia type I,spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,myotonic dystrophy, spongiform encephalopathies, hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eye disease,Sjogren's disease, Osteoporosis, Osteopenia, Gorham's Syndrome, chloridechannelopathies, myotonia congenita (Thomson and Becker forms),Bartter's syndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, PrimaryCiliary Dyskinesia (PCD), inherited disorders of the structure and/orfunction of cilia, PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus, or ciliary aplasia.

In another embodiment, the invention features a method of treating aCFTR mediated disease selected from cystic fibrosis, COPD, emphysema,dry-eye disease, or osteoporosis in a subject comprising administeringto the subject an effective amount of any of the above formulations ofCompound 1. In another embodiment, the CFTR mediated disease is cysticfibrosis.

In another embodiment, the method comprises administering an additionaltherapeutic agent. In another embodiment, the additional therapeuticagent is selected from a mucolytic agent, bronchodialator, ananti-biotic, an anti-infective agent, an anti-inflammatory agent, a CFTRmodulator other than Compound 1, or a nutritional agent.

In another aspect, the invention features a pharmaceutical pack or kitcomprising any of the above formulations of Compound 1 and instructionsfor use thereof.

Processes described herein can be used to prepare the compositions ofthis invention. The amounts and the features of the components used inthe processes would be as described herein.

DETAILED DESCRIPTION OF THE INVENTION Definitions

As used herein, the following definitions shall apply unless otherwiseindicated.

The term “CFTR” as used herein means cystic fibrosis transmembraneconductance regulator or a mutation thereof capable of regulatoractivity, including, but not limited to, ΔF508 CFTR and G551D CFTR (see,e.g., http://www.genet.sickkids.on.ca/cftr/, for CFTR mutations).

The term “modulating” as used herein means increasing or decreasing,e.g. activity, by a measurable amount.

Unless otherwise stated, structures depicted herein are also meant toinclude all isomeric (e.g., enantiomeric, diastereomeric, and geometric(or conformational)) forms of the structure; for example, the R and Sconfigurations for each asymmetric center, (Z) and (E) double bondisomers, and (Z) and (E) conformational isomers. Therefore, singlestereochemical isomers as well as enantiomeric, diastereomeric, andgeometric (or conformational) mixtures of the present compounds arewithin the scope of the invention. All tautomeric forms of the Compound1 are included herein. For example, Compound 1 may exist as tautomers,both of which are included herein:

Additionally, unless otherwise stated, structures depicted herein arealso meant to include compounds that differ only in the presence of oneor more isotopically enriched atoms. For example, Compound 1, whereinone or more hydrogen atoms are replaced deuterium or tritium, or one ormore carbon atoms are replaced by a ¹³C- or ¹⁴C-enriched carbon arewithin the scope of this invention. Such compounds are useful, forexample, as analytical tools, probes in biological assays, or compoundswith improved therapeutic profile.

The term “protecting group,” abbreviated as P, as used herein refers toany chemical group introduced into a molecule by chemical modificationof a functional group in order to obtain chemoselectivity in asubsequent chemical reaction. Non-limiting examples of alcoholprotecting groups include acetyl (Ac), benzoyl (Bz), benzyl (Bn),β-methoxyethoxymethyl ether (MEM), dimethoxytrityl (DMT), methoxymethylether (MOM), methoxytrityl (MMT), p-methoxybenzyl ether (PMB), pivaloyl(Piv), tetrahydropyranyl (THP), trityl (Tr), and trimethylsilyl (TMS).In one embodiment, the protecting group is Bn which has the structure—CH₂C₆H₅.

The abbreviation “DCM” stands for dichloromethane. The abbreviation“IPA” stands for isopropyl alcohol. The abbreviation “DMSO” stands fordimethylsulfoxide. The abbreviation “MTBE” stands for methyl t-butylether. The abbreviation “THF” stands for tetrahydrofuran. Theabbreviation “TEA” stands for triethylamine. The abbreviation “dba” asin Pd(dba)₂ stands for dibenzylideneacetone. The abbreviation “dppf” asin Pd(dppf)Cl₂ stands for stands for 1,1′-bis(diphenylphosphino)ferrocene.

Methods of Preparing Compound 1.

Compound 1 is(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide and, in one embodiment, is prepared by coupling an acidchloride moiety with an amine moiety according to Schemes 1-3.

Wherein compound 2 is prepared according to Scheme 2.

Wherein compound 3 is prepared according to Scheme 3.

Formulations of Compound 1 can be prepared according to the followingflow chart.

In one embodiment, formulations of Compound 1 can be prepared accordingto the following flow chart.

In one embodiment, the surfactant is added as an aqueous solutionprepared beforehand. In another embodiment, the concentration of theaqueous surfactant solution is about 20 to 25% by weight. In anotherembodiment, the concentration of the aqueous surfactant solution isabout 22% by weight. In another embodiment, the surfactant is vitamin ETPGS and the contration is about 22% by weight.

In another embodiment, the elevated temperature is about 30 to 50° C. Inanother embodiment, the elevated temperature is about 35 to 45° C. Inanother embodiment, the elevated temperature is about 40° C.

Formulations of Compound 1 may also be prepared by the following flowchart.

In another embodiment, formulations of Compound 1 can be preparedaccording to the following flow chart.

In this set of embodiments, Compound 1 and the surfactant, e.g. vitaminE TPGS, are dissolved in the PEG, e.g. PEG400, followed by dilution.Previously, the surfactant was added as an aqueous solution whichprovided the dilution.

In another embodiment, the elevated temperature is about 30 to 50° C. Inanother embodiment, the elevated temperature is about 35 to 45° C. Inanother embodiment, the elevated temperature is about 40° C.

The Compound 1 formulations of the invention demonstrate good in vivoexposure in dog studies. The PEG cosolvent and vitamin E TPGS inducedmicelle formation increase Compound 1 solubility from 7 micrograms toseveral milligrams per g of solution.

Uses, Formulation and Administration

Aqueous Formulations

In one aspect of the invention, aqueous formulations are providedcomprising Compound 1 as described herein, water, and a polyethyleneglycol, and optionally comprising other agents such as a taste maskerand/or flavorant, and additional pharmaceutically acceptable carriers,adjuvants or vehicles. In certain embodiments, these formulationsoptionally further comprise one or more additional therapeutic agents.

It will also be appreciated that Compound 1 can exist as apharmaceutically acceptable derivative or a prodrug thereof. Accordingto the invention, a pharmaceutically acceptable derivative or a prodrugincludes, but is not limited to esters, salts of such esters, or anyother adduct or derivative which upon administration to a patient inneed thereof is capable of providing, directly or indirectly, a compoundas otherwise described herein, or a metabolite or residue thereof.

1. Polyethylene Glycol

Polyethylene glycol (PEG) is a polyether compound and includespolyethylene oxide (PEO) and polyoxyethylene (POE). PEG, PEO, and POErefer to an oligomer or polymer of ethylene oxide. The three names arechemically synonymous, but historically PEG has tended to refer tooligomers and polymers with a molecular mass below 20,000 g/mol, PEO topolymers with a molecular mass above 20,000 g/mol, and POE to a polymerof any molecular mass. As used herein, PEG refers to a polyethyleneglycol of any molecular mass that is amenable for use in apharmaceutical formulations. Such suitable polyethylene glycols are wellknown in the art; see, e.g.,http://www.medicinescomplete.com/mc/excipients/current, which isincorporated herein by reference. Exemplary PEGs include low molecularweight PEGs such as PEG 200, PEG 300, PEG 400, etc. The number thatfollows the term “PEG” indicates the average molecular weight of thatparticular polymer. E.g., PEG 400 is a polyethylene glycol polymerwherein the average molecular weight of the polymer therein is about400.

In one embodiment, the PEG has an average molecular weight of from about200 to about 600. In another embodiment, PEG is PEG 400 (for example aPEG having a molecular weight of from about 380 to about 420 g/mol).

2. Surfactants

Surfactants reduce the interfacial tension between water and an organiccompound such as Compound 1 by adsorbing at the water-Compound 1interface, thus facilitating Compound 1 wettability and dissolution.Surfactants increase the solubility of Compound 1 via micelle formationand contribute to the stability of the aqueous solution. Surfactants areoften classified into four primary groups; anionic, cationic, non-ionic,and zwitterionic (dual charge). In a preferred embodiment, thesurfactant is an anionic, cationic, or nonionic surfactant.

Anionic surfactants may be chosen from salts of dodecyl sulfate, laurylsulfate, laureth sulfate, alkyl benzene sulfonates, butanoic acid,hexanoic acid, octanoic acid, decanoic acid, lauric acid, myristic acid,palmitic acid, stearic acid, arachidic acid, behenic acid, myristoleicacid, palmitoleic acid, oleic acid, linoleic acid, alpha-linolenic acid,arachidonic acid, eicosapentaenoic acid, erucic acid, or docosahexaenoicacid.

Cationic surfactants may be chosen from cetyl trimethylammonium bromide,cetylpyridinium chloride, polethoxylated tallow amine, benzalkoniumchloride, and benzethonium chloride.

Nonionic surfactants may be chosen from vitamine E derivatives,polysorbates, alkyl poly(ethylene oxide), poloxamine, alkylpolyglucosides, octyl glucoside, decyl maltoside, fatty alcohol, cetylalcohol, oleyl alcohol, cocamide MEA, cocamide DEA, and cocamide TEA. Inparticular, the nonionic surfactant is the vitamine E derivative vitaminE, d-α-tocopheryl polyethylene glycol 1000 succinate (vitamin E TPGS).Non-ionic surfactants also include the PEG derivatives under the brandname Solutol. For example Solutol HS 15 is prepared by reacting 15 molesof ethylene oxide with 1 mole of hydroxyl stearic acid.

The oral formulations of the invention generally comprise from about 5to about 15% by weight surfactant. In another embodiment, theconcentration of surfactant is from about 10 to about 12% by weight. Inanother embodiment, the concentration of surfactant is about 11% byweight.

3. Taste Masker and/or Flavoring Agent

As previously stated, it is advantageous to include a taste maskingagent in the oral formulations of Compound 1. Such taste masking agentsare alkali metal and alkaline earth metal chlorides including sodiumchloride, lithium chloride, potassium chloride, magnesium chloride, andcalcium chloride. Sodium chloride is preferred. The taste masking agentis generally included in the suspension in a taste-masking amount,generally an amount of about 0.5 to about 2.0 weight % as taste maskerbased on the weight of the suspension. For other salts, equivalent molaramounts can be calculated. Other taste maskers include sugars, with orwithout the presence of other sweetening and/or flavoring agents. Whenused, flavoring agents may be chosen from synthetic flavor oils andflavoring aromatics and/or natural oils, extracts from plant leaves,flowers, fruits, and so forth and combinations thereof. These mayinclude cinnamon oil, oil of wintergreen, peppermint oils, clove oil,bay oil, anise oil, eucalyptus, thyme oil, cedar leaf oil, oil ofnutmeg, oil of sage, oil of bitter almonds, and cassia oil. Also usefulas flavors are vanilla, citrus oil, including lemon, orange, grape, limeand grapefruit, and fruit essence, including apple, banana, pear, peach,strawberry, raspberry, cherry, plum, pineapple, apricot, and so forth.The amount of flavoring may depend on a number of factors including theorganoleptic effect desired. Generally the flavoring will be present inan amount of from about 0.01 to about 1.0 percent by weight based on thetotal suspension weight.

As described above, the formulations of the present invention cancomprise a pharmaceutically acceptable carrier, adjuvant, or vehicle,additional to water which, as used herein, includes any and allsolvents, diluents, or other liquid vehicle, dispersion or suspensionaids, surface active agents, isotonic agents, thickening or emulsifyingagents, preservatives, solid binders, lubricants and the like, as suitedto the particular dosage form desired. Remington's PharmaceuticalSciences, Sixteenth Edition, E. W. Martin (Mack Publishing Co., Easton,Pa., 1980) discloses various carriers used in formulatingpharmaceutically acceptable compositions and known techniques for thepreparation thereof. Except insofar as any conventional carrier mediumis incompatible with the compounds of the invention, such as byproducing any undesirable biological effect or otherwise interacting ina deleterious manner with any other component(s) of the pharmaceuticallyacceptable composition, its use is contemplated to be within the scopeof this invention. Some examples of materials which can serve aspharmaceutically acceptable carriers include, but are not limited to,ion exchangers, alumina, aluminum stearate, lecithin, serum proteins,such as human serum albumin, partial glyceride mixtures of saturatedvegetable fatty acids, salts or electrolytes, such as protamine sulfate,disodium hydrogen phosphate, potassium hydrogen phosphate, sodiumchloride, zinc salts, colloidal silica, magnesium trisilicate, polyvinylpyrrolidone, polyacrylates, waxes, polyethylene-polyoxypropylene-blockpolymers, wool fat, sugars such as lactose, glucose and sucrose;starches such as corn starch and potato starch; malt; gelatin; talc;excipients such as cocoa butter and suppository waxes; oils such aspeanut oil, cottonseed oil; safflower oil; sesame oil; olive oil; cornoil and soybean oil; glycols; such a propylene glycol or polyethyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; alginicacid; pyrogen-free water; isotonic saline; Ringer's solution; and ethylalcohol, as well as other non-toxic compatible lubricants such as sodiumlauryl sulfate and magnesium stearate, as well as coloring agents,releasing agents, coating agents, sweetening, perfuming agents,preservatives and antioxidants can also be present in the composition,according to the judgment of the formulator.

Uses of Compounds and Pharmaceutically Acceptable Compositions

In yet another aspect, the present invention provides a method oftreating a condition, disease, or disorder implicated by CFTR. Incertain embodiments, the present invention provides a method of treatinga condition, disease, or disorder implicated by a deficiency of CFTRactivity, the method comprising administering an oral formulationcomprising Compound 1 described herein to a subject, preferably amammal, in need thereof.

A “CFTR-mediated disease” as used herein is a disease selected fromcystic fibrosis, asthma, smoke induced COPD, chronic bronchitis,rhinosinusitis, constipation, pancreatitis, pancreatic insufficiency,male infertility caused by congenital bilateral absence of the vasdeferens (CBAVD), mild pulmonary disease, idiopathic pancreatitis,allergic bronchopulmonary aspergillosis (ABPA), liver disease,hereditary emphysema, hereditary hemochromatosis,coagulation-fibrinolysis deficiencies, protein C deficiency, Type 1hereditary angioedema, lipid processing deficiencies, familialhypercholesterolemia, Type 1 chylomicronemia, abetalipoproteinemia,lysosomal storage diseases, 1-cell disease/pseudo-Hurler,mucopolysaccharidoses, Sandhof/Tay-Sachs, Crigler-Najjar type II,polyendocrinopathy/hyperinsulemia, Diabetes mellitus, Laron dwarfism,myleoperoxidase deficiency, primary hypoparathyroidism, melanoma,glycanosis CDG type 1, congenital hyperthyroidism, osteogenesisimperfecta, hereditary hypofibrinogenemia, ACT deficiency, Diabetesinsipidus (DI), neurophyseal DI, neprogenic DI, Charcot-Marie Toothsyndrome, Perlizaeus-Merzbacher disease, neurodegenerative diseases,Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis,progressive supranuclear plasy, Pick's disease, several polyglutamineneurological disorders, Huntington's, spinocerebullar ataxia type I,spinal and bulbar muscular atrophy, dentatorubal pallidoluysian,myotonic dystrophy, spongiform encephalopathies, hereditaryCreutzfeldt-Jakob disease (due to prion protein processing defect),Fabry disease, Straussler-Scheinker syndrome, COPD, dry-eye disease,Sjogren's disease, Osteoporosis, Osteopenia, Gorham's Syndrome, chloridechannelopathies, myotonia congenita (Thomson and Becker forms),Bartter's syndrome type III, Dent's disease, hyperekplexia, epilepsy,hyperekplexia, lysosomal storage disease, Angelman syndrome, PrimaryCiliary Dyskinesia (PCD), inherited disorders of the structure and/orfunction of cilia, PCD with situs inversus (also known as Kartagenersyndrome), PCD without situs inversus, or ciliary aplasia.

In certain embodiments, the present invention provides a method oftreating a CFTR-mediated disease in a subject comprising the step ofadministering to the subject an effective amount of an oral formulationcomprising Compound 1 described herein.

According to another embodiment, the invention provides a method oftreating cystic fibrosis, emphysema, COPD, dry-eye disease, orosteoporosis in a subject comprising the step of administering to thesubject an oral formulation comprising Compound 1 described herein.

According to the invention an “effective amount” of an oral formulationof Compound 1 is that amount effective for treating or lessening theseverity of any of the diseases recited above.

In certain embodiments, an oral formulation of Compound 1 describedherein is useful for treating or lessening the severity of cysticfibrosis in subjects who exhibit residual CFTR activity in the apicalmembrane of respiratory and non-respiratory epithelia. The presence ofresidual CFTR activity at the epithelial surface can be readily detectedusing methods known in the art, e.g., standard electrophysiological,biochemical, or histochemical techniques. Such methods identify CFTRactivity using in vivo or ex vivo electrophysiological techniques,measurement of sweat or salivary Cl⁻ concentrations, or ex vivobiochemical or histochemical techniques to monitor cell surface density.Using such methods, residual CFTR activity can be readily detected inpatients heterozygous or homozygous for a variety of differentmutations, including patients homozygous or heterozygous for the mostcommon mutation, ΔF508.

In one embodiment, an oral formulation of Compound 1 described herein isuseful for treating or lessening the severity of cystic fibrosis inpatients within certain genotypes exhibiting residual CFTR activity,e.g., class III mutations (impaired regulation or gating), class IVmutations (altered conductance), or class V mutations (reducedsynthesis) (Lee R. Choo-Kang, Pamela L., Zeitlin, Type I, II, III, IV,and V cystic fibrosis Tansmembrane Conductance Regulator Defects andOpportunities of Therapy; Current Opinion in Pulmonary Medicine6:521-529, 2000). Other patient genotypes that exhibit residual CFTRactivity include patients homozygous for one of these classes orheterozygous with any other class of mutations, including class Imutations, class II mutations, or a mutation that lacks classification.

In one embodiment, an oral formulation of Compound 1 described herein isuseful for treating or lessening the severity of cystic fibrosis inpatients within certain clinical phenotypes, e.g., a moderate to mildclinical phenotype that typically correlates with the amount of residualCFTR activity in the apical membrane of epithelia. Such phenotypesinclude patients exhibiting pancreatic insufficiency or patientsdiagnosed with idiopathic pancreatitis and congenital bilateral absenceof the vas deferens, or mild lung disease.

The exact amount required will vary from subject to subject, dependingon the species, age, and general condition of the subject, the severityof the infection, the particular agent, its mode of administration, andthe like. The compounds of the invention are preferably formulated indosage unit form for ease of administration and uniformity of dosage.The expression “dosage unit form” as used herein refers to a physicallydiscrete unit of agent appropriate for the patient to be treated. Itwill be understood, however, that the total daily usage of the compoundsand compositions of the present invention will be decided by theattending physician within the scope of sound medical judgment. Thespecific effective dose level for any particular patient or organismwill depend upon a variety of factors including the disorder beingtreated and the severity of the disorder; the activity of the specificcompound employed; the specific composition employed; the age, bodyweight, general health, sex and diet of the patient; the time ofadministration, route of administration, and rate of excretion of thespecific compound employed; the duration of the treatment; drugs used incombination or coincidental with the specific compound employed, andlike factors well known in the medical arts. The terms “patient” and“subject” are used synonymously herein, and refer to an animal,preferably a mammal, and most preferably a human.

In certain embodiments, Compound 1 may be administered orally at dosagelevels of about 0.01 mg/kg to about 50 mg/kg and preferably from about 1mg/kg to about 25 mg/kg, of subject body weight per day, one or moretimes a day, to obtain the desired therapeutic effect.

In certain embodiments, the dosage amount of Compound 1 in the dosageunit form is from about 50 mg to about 2,000 mg. In another embodiment,the dosage amount of Compound 1 is from about 200 mg to about 900 mg. Inanother embodiment, the dosage amount of Compound 1 is from about 300 mgto about 800 mg. In another embodiment, the dosage amount of Compound 1is from about 400 mg to about 700 mg. In another embodiment, the dosageamount of Compound 1 is from about 500 mg to about 600 mg.

It will also be appreciated that the oral formulations of Compound 1described herein can be employed in combination therapies, that is, theoral formulations of Compound 1 can be administered concurrently with,prior to, or subsequent to, one or more other desired therapeutics ormedical procedures. The particular combination of therapies(therapeutics or procedures) to employ in a combination regimen willtake into account compatibility of the desired therapeutics and/orprocedures and the desired therapeutic effect to be achieved. It willalso be appreciated that the therapies employed may achieve a desiredeffect for the same disorder (for example, an inventive compound may beadministered concurrently with another agent used to treat the samedisorder), or they may achieve different effects (e.g., control of anyadverse effects). As used herein, additional therapeutic agents that arenormally administered to treat or prevent a particular disease, orcondition, are known as “appropriate for the disease, or condition,being treated”.

In one embodiment, the additional agent is selected from a mucolyticagent, bronchodialator, an anti-biotic, an anti-infective agent, ananti-inflammatory agent, a CFTR modulator other than a compound of thepresent invention, or a nutritional agent.

In one embodiment, the additional therapeutic agent is an antibiotic.Exemplary antibiotics useful herein include tobramycin, includingtobramycin inhaled powder (TIP), azithromycin, aztreonam, including theaerosolized form of aztreonam, amikacin, including liposomalformulations thereof, ciprofloxacin, including formulations thereofsuitable for administration by inhalation, levoflaxacin, includingaerosolized formulations thereof, and combinations of two antibiotics,e.g., fosfomycin and tobramycin.

In another embodiment, the additional agent is a mucolyte. Exemplarymucolytes useful herein includes Pulmozyme®.

In another embodiment, the additional agent is a bronchodialator.Exemplary bronchodialtors include albuterol, metaprotenerol sulfate,pirbuterol acetate, salmeterol, or tetrabuline sulfate.

In another embodiment, the additional agent is effective in restoringlung airway surface liquid. Such agents improve the movement of salt inand out of cells, allowing mucus in the lung airway to be more hydratedand, therefore, cleared more easily. Exemplary such agents includehypertonic saline, denufosol tetrasodium([[(3S,5R)-5-(4-amino-2-oxopyrimidin-1-yl)-3-hydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl][[[(2R,3S,4R,5R)-5-(2,4-dioxopyrimidin-1-yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl]oxy-hydroxyphosphoryl]hydrogen phosphate), or bronchitol (inhaled formulation of mannitol).

In another embodiment, the additional agent is an anti-inflammatoryagent, i.e., an agent that can reduce the inflammation in the lungs.Exemplary such agents useful herein include ibuprofen, docosahexanoicacid (DHA), sildenafil, inhaled glutathione, pioglitazone,hydroxychloroquine, or simavastatin.

In another embodiment, the additional agent is a CFTR modulator otherthan Compound 1, i.e., an agent that has the effect of modulating CFTRactivity. Exemplary such agents include ataluren (“PTC124®”;3-[5-(2-fluorophenyl)-1,2,4-oxadiazol-3-yl]benzoic acid), sinapultide,lancovutide, depelestat (a human recombinant neutrophil elastaseinhibitor), cobiprostone(7-{(2R,4aR,5R,7aR)-2-[(3S)-1,1-difluoro-3-methylpentyl]-2-hydroxy-6-oxooctahydrocyclopenta[b]pyran-5-yl}heptanoicacid), andN-(5-hydroxy-2,4-ditert-butyl-phenyl)-4-oxo-1H-quinoline-3-carboxamide.

In another embodiment, the additional agent is a nutritional agent.Exemplary nutritional agents include pancrelipase (pancreating enzymereplacement), including Pancrease®, Pancreacarb®, Ultrase®, or Creon®,Liprotomase® (formerly Trizytek®), Aquadeks®, or glutathione inhalation.In one embodiment, the additional nutritional agent is pancrelipase.

In another embodiment, the additional agent is a compound selected fromgentamicin, curcumin, cyclophosphamide, 4-phenylbutyrate, miglustat,felodipine, nimodipine, Philoxin B, geniestein, Apigenin, cAMP/cGMPmodulators such as rolipram, sildenafil, milrinone, tadalafil, aminone,isoproterenol, albuterol, and almeterol, deoxyspergualin, HSP 90inhibitors, HSP 70 inhibitors, proteosome inhibitors such as epoxomicin,lactacystin, etc.

In another embodiment, the additional agent is a compound disclosed inWO 2004028480, WO 2004110352, WO 2005094374, WO 2005120497, or WO2006101740.

In another embodiment, the additional agent is a benzo[c]quinoliziniumderivative that exhibits CFTR modulation activity or a benzopyranderivative that exhibits CFTR modulation activity.

In another embodiment, the additional agent is a compound disclosed inU.S. Pat. No. 7,202,262, U.S. Pat. No. 6,992,096, US20060148864,US20060148863, US20060035943, US20050164973, WO2006110483, WO2006044456,WO2006044682, WO2006044505, WO2006044503, WO2006044502, or WO2004091502.

In another embodiment, the additional agent is a compound disclosed inWO2004080972, WO2004111014, WO2005035514, WO2005049018, WO2006099256,WO2006127588, or WO2007044560.

These combinations are useful for treating the diseases described hereinincluding cystic fibrosis. These combinations are also useful in thekits described herein.

The amount of additional therapeutic agent present in the compositionsof this invention will be no more than the amount that would normally beadministered in a composition comprising that therapeutic agent as theonly active agent. Preferably the amount of additional therapeutic agentin the presently disclosed compositions will range from about 50% to100% of the amount normally present in a composition comprising thatagent as the only therapeutically active agent.

In order that the invention described herein may be more fullyunderstood, the following examples are set forth. It should beunderstood that these examples are for illustrative purposes only andare not to be construed as limiting this invention in any manner.

EXAMPLES Methods & Materials

Vitride® (sodium bis(2-methoxyethoxy)aluminum hydride [orNaAlH₂(OCH₂CH₂OCH₃)₂], 65 wgt % solution in toluene) was purchased fromAldrich Chemicals. 3-Fluoro-4-nitroaniline was purchased from CapotChemicals. 5-Bromo-2,2-difluoro-1,3-benzodioxole was purchased from AlfaAesar. 2,2-Difluoro-1,3-benzodioxole-5-carboxylic acid was purchasedfrom Saltigo (an affiliate of the Lanxess Corporation).

Anywhere in the present application where a name of a compound may notcorrectly describe the structure of the compound, the structuresupersedes the name and governs.

Synthesis of Compound 1

Acid Moiety Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile

A reactor was purged with nitrogen and charged with 900 mL of toluene.The solvent was degassed via nitrogen sparge for no less than 16 h. Tothe reactor was then charged Na₃PO₄ (155.7 g, 949.5 mmol), followed bybis(dibenzylideneacetone) palladium (0) (7.28 g, 12.66 mmol). A 10% w/wsolution of tert-butylphosphine in hexanes (51.23 g, 25.32 mmol) wascharged over 10 min at 23° C. from a nitrogen purged addition funnel.The mixture was allowed to stir for 50 min, at which time5-bromo-2,2-difluoro-1,3-benzodioxole (75 g, 316.5 mmol) was added over1 min. After stirring for an additional 50 min, the mixture was chargedwith ethyl cyanoacetate (71.6 g, 633.0 mmol) over 5 min followed bywater (4.5 mL) in one portion. The mixture was heated to 70° C. over 40min and analyzed by HPLC every 1-2 h for the percent conversion of thereactant to the product. After complete conversion was observed(typically 100% conversion after 5-8 h), the mixture was cooled to20-25° C. and filtered through a celite pad. The celite pad was rinsedwith toluene (2×450 mL) and the combined organics were concentrated to300 mL under vacuum at 60-65° C. The concentrate was charged with 225 mLDMSO and concentrated under vacuum at 70-80° C. until activedistillation of the solvent ceased. The solution was cooled to 20-25° C.and diluted to 900 mL with DMSO in preparation for Step 2. ¹H NMR (500MHz, CDCl₃) δ 7.16-7.10 (m, 2H), 7.03 (d, J=8.2 Hz, 1H), 4.63 (s, 1H),4.19 (m, 2H), 1.23 (t, J=7.1 Hz, 3H).

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

The DMSO solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-1-ethylacetate-acetonitrile fromabove was charged with 3 N HCl (617.3 mL, 1.85 mol) over 20 min whilemaintaining an internal temperature <40° C. The mixture was then heatedto 75° C. over 1 h and analyzed by HPLC every 1-2 h for % conversion.When a conversion of >99% was observed (typically after 5-6 h), thereaction was cooled to 20-25° C. and extracted with MTBE (2×525 mL),with sufficient time to allow for complete phase separation during theextractions. The combined organic extracts were washed with 5% NaCl(2×375 mL). The solution was then transferred to equipment appropriatefor a 1.5-2.5 Torr vacuum distillation that was equipped with a cooledreceiver flask. The solution was concentrated under vacuum at <60° C. toremove the solvents. (2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrilewas then distilled from the resulting oil at 125-130° C. (oventemperature) and 1.5-2.0 Torr.(2,2-Difluoro-1,3-benzodioxol-5-yl)-acetonitrile was isolated as a clearoil in 66% yield from 5-bromo-2,2-difluoro-1,3-benzodioxole (2 steps)and with an HPLC purity of 91.5% AUC (corresponds to a w/w assay of95%). ¹H NMR (500 MHz, DMSO) δ 7.44 (br s, 1H), 7.43 (d, J=8.4 Hz, 1H),7.22 (dd, J=8.2, 1.8 Hz, 1H), 4.07 (s, 2H).

Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile

A stock solution of 50% w/w NaOH was degassed via nitrogen sparge for noless than 16 h. An appropriate amount of MTBE was similarly degassed forseveral hours. To a reactor purged with nitrogen was charged degassedMTBE (143 mL) followed by(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (40.95 g, 207.7 mmol)and tetrabutylammonium bromide (2.25 g, 10.38 mmol). The volume of themixture was noted and the mixture was degassed via nitrogen sparge for30 min. Enough degassed MTBE is charged to return the mixture to theoriginal volume prior to degassing. To the stirring mixture at 23.0° C.was charged degassed 50% w/w NaOH (143 mL) over 10 min followed by1-bromo-2-chloroethane (44.7 g, 311.6 mmol) over 30 min. The reactionwas analyzed by HPLC in 1 h intervals for % conversion. Before sampling,stirring was stopped and the phases allowed to separate. The top organicphase was sampled for analysis. When a conversion>99% was observed(typically after 2.5-3 h), the reaction mixture was cooled to 10° C. andwas charged with water (461 mL) at such a rate as to maintain atemperature <25° C. The temperature was adjusted to 20-25° C. and thephases separated. Note: sufficient time should be allowed for completephase separation. The aqueous phase was extracted with MTBE (123 mL),and the combined organic phase was washed with 1 N HCl (163 mL) and 5%NaCl (163 mL). The solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in MTBE wasconcentrated to 164 mL under vacuum at 40-50° C. The solution wascharged with ethanol (256 mL) and again concentrated to 164 mL undervacuum at 50-60° C. Ethanol (256 mL) was charged and the mixtureconcentrated to 164 mL under vacuum at 50-60° C. The resulting mixturewas cooled to 20-25° C. and diluted with ethanol to 266 mL inpreparation for the next step. ¹H NMR (500 MHz, DMSO) δ 7.43 (d, J=8.4Hz, 1H), 7.40 (d, J=1.9 Hz, 1H), 7.30 (dd, J=8.4, 1.9 Hz, 1H), 1.75 (m,2H), 1.53 (m, 2H).

Synthesis of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid

The solution of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile in ethanolfrom the previous step was charged with 6 N NaOH (277 mL) over 20 minand heated to an internal temperature of 77-78° C. over 45 min. Thereaction progress was monitored by HPLC after 16 h. Note: theconsumption of both(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile and theprimary amide resulting from partial hydrolysis of(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarbonitrile weremonitored. When a % conversion>99% was observed (typically 100%conversion after 16 h), the reaction mixture was cooled to 25° C. andcharged with ethanol (41 mL) and DCM (164 mL). The solution was cooledto 10° C. and charged with 6 N HCl (290 mL) at such a rate as tomaintain a temperature <25° C. After warming to 20-25° C., the phaseswere allowed to separate. The bottom organic phase was collected and thetop aqueous phase was back extracted with DCM (164 mL). Note: theaqueous phase was somewhat cloudy before and after the extraction due toa high concentration of inorganic salts. The organics were combined andconcentrated under vacuum to 164 mL. Toluene (328 mL) was charged andthe mixture condensed to 164 mL at 70-75° C. The mixture was cooled to45° C., charged with MTBE (364 mL) and stirred at 60° C. for 20 min. Thesolution was cooled to 25° C. and polish filtered to remove residualinorganic salts. MTBE (123 mL) was used to rinse the reactor and thecollected solids. The combined organics were transferred to a cleanreactor in preparation for the next step.

Isolation of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid.

The solution of1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid fromthe previous step is concentrated under vacuum to 164 mL, charged withtoluene (328 mL) and concentrated to 164 mL at 70-75° C. The mixture wasthen heated to 100-105° C. to give a homogeneous solution. Afterstirring at that temperature for 30 min, the solution was cooled to 5°C. over 2 hours and maintained at 5° C. for 3 hours. The mixture wasthen filtered and the reactor and collected solid washed with cold 1:1toluene/n-heptane (2×123 mL). The material was dried under vacuum at 55°C. for 17 hours to provide1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid as anoff-white crystalline solid.1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid wasisolated in 79% yield from(2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (3 steps includingisolation) and with an HPLC purity of 99.0% AUC. ESI-MS m/z calc.242.04. found 241.58 (M+1)⁺; ¹H NMR (500 MHz, DMSO) δ 12.40 (s, 1H),7.40 (d, J=1.6 Hz, 1H), 7.30 (d, J=8.3 Hz, 1H), 7.17 (dd, J=8.3, 1.7 Hz,1H), 1.46 (m, 2H), 1.17 (m, 2H).

Alternative Synthesis of the Acid Moiety Synthesis of(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol

Commercially available 2,2-difluoro-1,3-benzodioxole-5-carboxylic acid(1.0 eq) is slurried in toluene (10 vol). Vitride® (2 eq) is added viaaddition funnel at a rate to maintain the temperature at 15-25° C. Atthe end of addition the temperature is increased to 40° C. for 2 h then10% (w/w) aq. NaOH (4.0 eq) is carefully added via addition funnelmaintaining the temperature at 40-50° C. After stirring for anadditional 30 minutes, the layers are allowed to separate at 40° C. Theorganic phase is cooled to 20° C. then washed with water (2×1.5 vol),dried (Na₂SO₄), filtered, and concentrated to afford crude(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol that is used directly inthe next step.

Synthesis of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole

(2,2-difluoro-1,3-benzodioxol-5-yl)-methanol (1.0 eq) is dissolved inMTBE (5 vol). A catalytic amount of DMAP (1 mol %) is added and SOCl₂(1.2 eq) is added via addition funnel. The SOCl₂ is added at a rate tomaintain the temperature in the reactor at 15-25° C. The temperature isincreased to 30° C. for 1 hour then cooled to 20° C. then water (4 vol)is added via addition funnel maintaining the temperature at less than30° C. After stirring for an additional 30 minutes, the layers areallowed to separate. The organic layer is stirred and 10% (w/v) aq. NaOH(4.4 vol) is added. After stirring for 15 to 20 minutes, the layers areallowed to separate. The organic phase is then dried (Na₂SO₄), filtered,and concentrated to afford crude5-chloromethyl-2,2-difluoro-1,3-benzodioxole that is used directly inthe next step.

Synthesis of (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile

A solution of 5-chloromethyl-2,2-difluoro-1,3-benzodioxole (1 eq) inDMSO (1.25 vol) is added to a slurry of NaCN (1.4 eq) in DMSO (3 vol)maintaining the temperature between 30-40° C. The mixture is stirred for1 hour then water (6 vol) is added followed by MTBE (4 vol). Afterstirring for 30 min, the layers are separated. The aqueous layer isextracted with MTBE (1.8 vol). The combined organic layers are washedwith water (1.8 vol), dried (Na₂SO₄), filtered, and concentrated toafford crude (2,2-difluoro-1,3-benzodioxol-5-yl)-acetonitrile (95%) thatis used directly in the next step.

The remaining steps are the same as described above for the synthesis ofthe acid moiety.

Amine Moiety Synthesis of 2-bromo-5-fluoro-4-nitroaniline

A flask was charged with 3-fluoro-4-nitroaniline (1.0 equiv) followed byethyl acetate (10 vol) and stirred to dissolve all solids.N-Bromosuccinimide (1.0 equiv) was added as a portion-wise as tomaintain internal temperature of 22° C. At the end of the reaction, thereaction mixture was concentrated in vacuo on a rotavap. The residue wasslurried in distilled water (5 vol) to dissolve and remove succinimide.(The succinimide can also be removed by water workup procedure.) Thewater was decanted and the solid was slurried in 2-propanol (5 vol)overnight. The resulting slurry was filtered and the wetcake was washedwith 2-propanol, dried in vacuum oven at 50° C. overnight with N₂ bleeduntil constant weight was achieved. A yellowish tan solid was isolated(50% yield, 97.5% AUC). Other impurities were a bromo-regioisomer (1.4%AUC) and a di-bromo adduct (1.1% AUC). ¹H NMR (500 MHz, DMSO)

8.19 (1H, d, J=8.1 Hz), 7.06 (br. s, 2H), 6.64 (d, 1H, J=14.3 Hz).

Synthesis of p-toluenesulfonic acid salt of(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol

A thoroughly dried flask under N₂ was charged with the following:Activated powdered 4 A molecular sieves (50 wt % based on2-bromo-5-fluoro-4-nitroaniline), 2-Bromo-5-fluoro-4-nitroaniline (1.0equiv), zinc perchlorate dihydrate (20 mol %), and toluene (8 vol). Themixture was stirred at room temperature for NMT 30 min. Lastly,(R)-benzyl glycidyl ether (2.0 equiv) in toluene (2 vol) was added in asteady stream. The reaction was heated to 80° C. (internal temperature)and stirred for approximately 7 hours or until2-Bromo-5-fluoro-4-nitroaniline was <5% AUC.

The reaction was cooled to room temperature and Celite (50 wt %) wasadded, followed by ethyl acetate (10 vol). The resulting mixture wasfiltered to remove Celite and sieves and washed with ethyl acetate (2vol). The filtrate was washed with ammonium chloride solution (4 vol,20% w/v). The organic layer was washed with sodium bicarbonate solution(4 vol×2.5% w/v). The organic layer was concentrated in vacuo on arotovap. The resulting slurry was dissolved in isopropyl acetate (10vol) and this solution was transferred to a Buchi hydrogenator.

The hydrogenator was charged with 5 wt % Pt(S)/C (1.5 mol %) and themixture was stirred under N₂ at 30° C. (internal temperature). Thereaction was flushed with N₂ followed by hydrogen. The hydrogenatorpressure was adjusted to 1 Bar of hydrogen and the mixture was stirredrapidly (>1200 rpm). At the end of the reaction, the catalyst wasfiltered through a pad of Celite and washed with dichloromethane (10vol). The filtrate was concentrated in vacuo. Any remaining isopropylacetate was chased with dichloromethane (2 vol) and concentrated on arotavap to dryness.

The resulting residue was dissolved in dichloromethane (10 vol).p-Toluenesulfonic acid monohydrate (1.2 equiv) was added and stirredovernight. The product was filtered and washed with dichloromethane (2vol) and suction dried. The wetcake was transferred to drying trays andinto a vacuum oven and dried at 45° C. with N₂ bleed until constantweight was achieved. p-Toluenesulfonic acid salt of(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-olwas isolated as an off-white solid.

Chiral purity was determined to be >97% ee.

Synthesis of (3-Chloro-3-methylbut-1-ynyl)trimethylsilane

Propargyl alcohol (1.0 equiv) was charged to a vessel. Aqueoushydrochloric acid (37%, 3.75 vol) was added and stirring begun. Duringdissolution of the solid alcohol, a modest endotherm (5-6° C.) isobserved. The resulting mixture was stirred overnight (16 h), slowlybecoming dark red. A 30 L jacketed vessel is charged with water (5 vol)which is then cooled to 10° C. The reaction mixture is transferredslowly into the water by vacuum, maintaining the internal temperature ofthe mixture below 25° C. Hexanes (3 vol) is added and the resultingmixture is stirred for 0.5 h. The phases were settled and the aqueousphase (pH<1) was drained off and discarded. The organic phase wasconcentrated in vacuo using a rotary evaporator, furnishing the productas red oil.

Synthesis of (4-(Benzyloxy)-3,3-dimethylbut-1-ynyl)trimethylsilane

Method A

All equivalent and volume descriptors in this part are based on a 250 greaction. Magnesium turnings (69.5 g, 2.86 mol, 2.0 equiv) were chargedto a 3 L 4-neck reactor and stirred with a magnetic stirrer undernitrogen for 0.5 h. The reactor was immersed in an ice-water bath. Asolution of the propargyl chloride (250 g, 1.43 mol, 1.0 equiv) in THF(1.8 L, 7.2 vol) was added slowly to the reactor, with stirring, untilan initial exotherm (˜10° C.) was observed. The Grignard reagentformation was confirmed by IPC using ¹H-NMR spectroscopy. Once theexotherm subsided, the remainder of the solution was added slowly,maintaining the batch temperature <15° C. The addition required ˜3.5 h.The resulting dark green mixture was decanted into a 2 L capped bottle.

All equivalent and volume descriptors in this part are based on a 500 greaction. A 22 L reactor was charged with a solution of benzylchloromethyl ether (95%, 375 g, 2.31 mol, 0.8 equiv) in THF (1.5 L, 3vol). The reactor was cooled in an ice-water bath. Two Grignard reagentbatches prepared as described above were combined and then added slowlyto the benzyl chloromethyl ether solution via an addition funnel,maintaining the batch temperature below 25° C. The addition required 1.5h. The reaction mixture was stirred overnight (16 h).

All equivalent and volume descriptors in this part are based on a 1 kgreaction. A solution of 15% ammonium chloride was prepared in a 30 Ljacketed reactor (1.5 kg in 8.5 kg of water, 10 vol). The solution wascooled to 5° C. Two Grignard reaction mixtures prepared as describedabove were combined and then transferred into the ammonium chloridesolution via a header vessel. An exotherm was observed in this quench,which was carried out at a rate such as to keep the internal temperaturebelow 25° C. Once the transfer was complete, the vessel jackettemperature was set to 25° C. Hexanes (8 L, 8 vol) was added and themixture was stirred for 0.5 h. After settling the phases, the aqueousphase (pH 9) was drained off and discarded. The remaining organic phasewas washed with water (2 L, 2 vol). The organic phase was concentratedin vacuo using a 22 L rotary evaporator, providing the crude product asan orange oil.

Method B

Magnesium turnings (106 g, 4.35 mol, 1.0 eq) were charged to a 22 Lreactor and then suspended in THF (760 mL, 1 vol). The vessel was cooledin an ice-water bath such that the batch temperature reached 2° C. Asolution of the propargyl chloride (760 g, 4.35 mol, 1.0 equiv) in THF(4.5 L, 6 vol) was added slowly to the reactor. After 100 mL was added,the addition was stopped and the mixture stirred until a 13° C. exothermwas observed, indicating the Grignard reagent initiation. Once theexotherm subsided, another 500 mL of the propargyl chloride solution wasadded slowly, maintaining the batch temperature <20° C. The Grignardreagent formation was confirmed by IPC using ¹H-NMR spectroscopy. Theremainder of the propargyl chloride solution was added slowly,maintaining the batch temperature <20° C. The addition required ˜1.5 h.The resulting dark green solution was stirred for 0.5 h. The Grignardreagent formation was confirmed by IPC using ¹H-NMR spectroscopy. Neatbenzyl chloromethyl ether was charged to the reactor addition funnel andthen added dropwise into the reactor, maintaining the batch temperaturebelow 25° C. The addition required 1.0 h. The reaction mixture wasstirred overnight. The aqueous work-up and concentration was carried outusing the same procedure and relative amounts of materials as in MethodA to give the product as an orange oil.

Synthesis of 4-Benzyloxy-3,3-dimethylbut-1-yne

A 30 L jacketed reactor was charged with methanol (6 vol) which was thencooled to 5° C. Potassium hydroxide (85%, 1.3 equiv) was added to thereactor. A 15-20° C. exotherm was observed as the potassium hydroxidedissolved. The jacket temperature was set to 25° C. A solution of4-benzyloxy-3,3-dimethyl-1-trimethylsilylbut-1-yne (1.0 equiv) inmethanol (2 vol) was added and the resulting mixture was stirred untilreaction completion, as monitored by HPLC. Typical reaction time at 25°C. is 3-4 h. The reaction mixture is diluted with water (8 vol) and thenstirred for 0.5 h. Hexanes (6 vol) was added and the resulting mixturewas stirred for 0.5 h. The phases were allowed to settle and then theaqueous phase (pH 10-11) was drained off and discarded. The organicphase was washed with a solution of KOH (85%, 0.4 equiv) in water (8vol) followed by water (8 vol). The organic phase was then concentrateddown using a rotary evaporator, yielding the title material as ayellow-orange oil. Typical purity of this material is in the 80% rangewith primarily a single impurity present. ¹H NMR (400 MHz, C₆D₆) δ 7.28(d, 2H, J=7.4 Hz), 7.18 (t, 2H, J=7.2 Hz), 7.10 (d, 1H, J=7.2 Hz), 4.35(s, 2H), 3.24 (s, 2H), 1.91 (s, 1H), 1.25 (s, 6H).

Synthesis ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleMethod A Synthesis of(R)-1-((4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl)-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-ol

p-Toluenesulfonic acid salt of(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-olwas freebased by stirring the solid in dichloromethane (5 vol) andsaturated NaHCO₃ solution (5 vol) until clear organic layer wasachieved. The resulting layers were separated and the organic layer waswashed with saturated NaHCO₃ solution (5 vol) followed by brine andconcentrated in vacuo to obtain(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-olfree base as an oil.

Palladium acetate (0.01 eq), dppb (0.015 eq), CuI (0.015 eq) andpotassium carbonate (3 eq) are suspended in acetonitrile (1.2 vol).After stirring for 15 minutes, a solution of4-benzyloxy-3,3-dimethylbut-1-yne (1.1 eq) in acetonitrile (0.2 vol) isadded. The mixture is sparged with nitrogen gas for 1 h and then asolution of(R)-1-((4-amino-2-bromo-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-olfree base (1 eq) in acetonitrile (4.1 vol) is added. The mixture issparged with nitrogen gas for another hour and then is heated to 80° C.Reaction progress is monitored by HPLC and the reaction is usuallycomplete within 3-5 h. The mixture is cooled to room temperature andthen filtered through Celite. The cake is washed with acetonitrile (4vol). The combined filtrates are azeotroped to dryness and then themixture is polish filtered into the next reactor. The acetonitrilesolution of(R)-1-((4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl)-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-olthus obtained is used directly in the next procedure (cyclization)without further manipulation.

Synthesis ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindole

Bis-acetonitriledichloropalladium (0.1 eq) and CuI (0.1 eq) are chargedto the reactor and then suspended in a solution of(R)-1-((4-amino-2-(4-(benzyloxy)-3,3-dimethylbut-1-yn-1-yl)-5-fluorophenyl)amino)-3-(benzyloxy)propan-2-olobtained above (1 eq) in acetonitrile (9.5 vol total). The mixture issparged with nitrogen gas for 1 h and then is heated to 80° C. Thereaction progress is monitored by HPLC and the reaction is typicallycomplete within 1-3 h. The mixture is filtered through Celite and thecake is washed with acetonitrile. A solvent swap into ethyl acetate (7.5vol) is performed. The ethyl acetate solution is washed with aqueousNH₃—NH₄Cl solution (2×2.5 vol) followed by 10% brine (2.5 vol). Theethyl acetate solution is then stirred with silica gel (1.8 wt eq) andSi-TMT (0.1 wt eq) for 6 h. After filtration, the resulting solution isconcentrated down. The residual oil is dissolved in DCM/heptane (4 vol)and then purified by column chromatography. The oil thus obtained isthen crystallized from 25% EtOAc/heptane (4 vol). Crystalline(R)-1-(5-amino-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-1-yl)-3-(benzyloxy)propan-2-olis typically obtained in 27-38% yield. ¹H NMR (400 MHz, DMSO) δ7.38-7.34 (m, 4H), 7.32-7.23 (m, 6H), 7.21 (d, 1H, J=12.8 Hz), 6.77 (d,1H, J=9.0 Hz), 6.06 (s, 1H), 5.13 (d, 1H, J=4.9 Hz), 4.54 (s, 2H), 4.46(br. s, 2H), 4.45 (s, 2H), 4.33 (d, 1H, J=12.4 Hz), 4.09-4.04 (m, 2H),3.63 (d, 1H, J=9.2 Hz), 3.56 (d, 1H, J=9.2 Hz), 3.49 (dd, 1H, J=9.8, 4.4Hz), 3.43 (dd, 1H, J=9.8, 5.7 Hz), 1.40 (s, 6H).

Synthesis ofN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleMethod B

Palladium acetate (33 g, 0.04 eq), dppb (94 g, 0.06 eq), and potassiumcarbonate (1.5 kg, 3.0 eq) are charged to a reactor. The free based oilbenzylglocolated 4-ammonium-2-bromo-5-fluoroaniline (1.5 kg, 1.0 eq) wasdissolved in acetonitrile (8.2 L, 4.1 vol) and then added to thereactor. The mixture was sparged with nitrogen gas for NLT 1 h. Asolution of 4-benzyloxy-3,3-dimethylbut-1-yne (70%, 1.1 kg, 1.05 eq) inacetonitrile was added to the mixture which was then sparged withnitrogen gas for NLT 1 h. The mixture was heated to 80° C. and thenstirred overnight. IPC by HPLC is carried out and the reaction isdetermined to be complete after 16 h. The mixture was cooled to ambienttemperature and then filtered through a pad of Celite (228 g). Thereactor and Celite pad were washed with acetonitrile (2×2 L, 2 vol). Thecombined phases are concentrated on a 22 L rotary evaporator until 8 Lof solvent have been collected, leaving the crude product in 7 L (3.5vol) of acetonitrile.

Bis-acetonitriledichloropalladium (144 g, 0.15 eq) was charged to thereactor. The crude solution was transferred back into the reactor andthe roto-vap bulb was washed with acetonitrile (4 L, 2 vol). Thecombined solutions were sparged with nitrogen gas for NLT 1 h. Thereaction mixture was heated to 80° C. for NLT 16 h. In process controlby HPLC shows complete consumption of starting material. The reactionmixture was filtered through Celite (300 g). The reactor and filter cakewere washed with acetonitrile (3 L, 1.5 vol). The combined filtrateswere concentrated to an oil by rotary evaporation. The oil was dissolvedin ethyl acetate (8.8 L, 4.4 vol). The solution was washed with 20%ammonium chloride (5 L, 2.5 vol) followed by 5% brine (5 L, 2.5 vol).Silica gel (3.5 kg, 1.8 wt. eq.) of silica gel was added to the organicphase, which was stirred overnight. Deloxan THP II metal scavenger (358g) and heptane (17.6 L) were added and the resulting mixture was stirredfor NLT 3 h. The mixture was filtered through a sintered glass funnel.The filter cake was washed with 30% ethyl acetate in heptane (25 L). Thecombined filtrates were concentrated under reduced pressure to giveN-benzylglycolated-5-amino-2-(2-benzyloxy-1,1-dimethylethyl)-6-fluoroindoleas a brown paste (1.4 kg).

Synthesis of Compound 1

Synthesis of Benzyl Protected Compound 1.

1-(2,2-Difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid (1.3equiv) was slurried in toluene (2.5 vol, based on1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid).Thionyl chloride (SOCl₂, 1.7 equiv) was added via addition funnel andthe mixture was heated to 60° C. The resulting mixture was stirred for 2h. The toluene and the excess SOCl2 were distilled off using rotavop.Additional toluene (2.5 vol, based on1-(2,2-difluoro-1,3-benzodioxol-5-yl)-cyclopropanecarboxylic acid) wasadded and the mixture was distilled down to 1 vol of toluene. A solutionof(R)-1-(5-amino-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-1-yl)-3-(benzyloxy)propan-2-ol(1 eq) and triethylamine (3 eq) in DCM (4 vol) is cooled to 0° C. Theacid chloride solution in toluene (1 vol) is added while maintaining thebatch temperature below 10° C. The reaction progress is monitored byHPLC, and the reaction is usually complete within minutes. After warmingto 25° C., the reaction mixture is washed with 5% NaHCO₃ (3.5 vol), 1 MNaOH (3.5 vol) and 1 M HCl (5 vol). A solvent swap to into methanol (2vol) is performed and the resulting solution of(R)—N-(1-(3-(benzyloxy)-2-hydroxypropyl)-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-5-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamidein methanol is used without further manipulation in the next step(hydrogenolysis).

Synthesis of Compound 1.

5% palladium on charcoal (˜50% wet, 0.01 eq) is charged to anappropriate hydrogenation vessel. The(R)—N-(1-(3-(benzyloxy)-2-hydroxypropyl)-2-(1-(benzyloxy)-2-methylpropan-2-yl)-6-fluoro-1H-indol-5-yl)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)cyclopropanecarboxamidesolution in methanol (2 vol) obtained above is added carefully, followedby a 3 M solution of HCl in methanol. The vessel is purged with nitrogengas and then with hydrogen gas. The mixture is stirred vigorously untilthe reaction is complete, as determined by HPLC analysis. Typicalreaction time is 3-5 h. The reaction mixture is filtered through Celiteand the cake is washed with methanol (2 vol). A solvent swap intoisopropanol (3 vol) is performed. Crude VX-661 is crystallized from 75%IPA-heptane (4 vol, ie. 1 vol heptane added to the 3 vol of IPA) and theresulting crystals are matured in 50% IPA-heptane (ie. 2 vol of heptaneadded to the mixture). Typical yields of compound 4 from the two-stepacylation/hydrogenolysis procedure range from 68% to 84%. Compound 4 canbe recrystallized from IPA-heptane following the same procedure justdescribed.

Compound 1 may also be prepared by one of several synthetic routesdisclosed in US published patent application US20090131492, incorporatedherein by reference.

Table 10 below recites analytical data for Compound 1.

TABLE 10 LC/ MS LC/ Cmpd. M + RT No. 1 min NMR 1 521.5 1.69 1H NMR(400.0 MHz, CD₃CN) d 7.69 (d, J = 7.7 Hz, 1H), 7.44 (d, J = 1.6 Hz, 1H),7.39 (dd, J = 1.7, 8.3 Hz, 1H), 7.31 (s, 1H), 7.27 (d, J = 8.3 Hz, 1H),7.20 (d, J = 12.0 Hz, 1H), 6.34 (s, 1H), 4.32 (d, J = 6.8 Hz, 2H),4.15-4.09 (m, 1H), 3.89 (dd, J = 6.0, 11.5 Hz, 1H), 3.63-3.52 (m, 3H),3.42 (d, J = 4.6 Hz, 1H), 3.21 (dd, J = 6.2, 7.2 Hz, 1H), 3.04 (t, J =5.8 Hz, 1H), 1.59 (dd, J = 3.8, 6.8 Hz, 2H), 1.44 (s, 3H), 1.33 (s, 3H)and 1.18 (dd, J = 3.7, 6.8 Hz, 2H) ppm.

Preparation of Compound 1 Formulations

Preparation of a 40 g Formulation Comprising 50 mg of Compound 1

A 40 g aqueous formulation is prepared with the following components andamounts:

Amount Component (g) Compound 1 0.05 PEG 400 19.95 Vitamin E TPGS 4.48Water 15.52

A 22.4% by weight vitamin E TPGS solution is prepared separately byadding vitamin E TPGS (4.48 g) to a clear glass jar equipped with amagnetic stirring bar. Water (15.52 mL) is added and the jar is sealedand stirred at a rate such that the vortex depth is approximately ⅔ ofthe liquid depth. Stir the mixture of vitamin E TPGS and water at roomtemperature until all of the vitamin E TPGS is dissolved and there areno visible solid particles. Depending on the size of the vitamin E TPGSsolid particles, this step may take more than 4 hours. Stirring of thesolution in the closed container can be done overnight. To speed up thevitamin E TPGS dissolution process, it can be carried out in a waterbath at 30-35° C. Remove the solution from stirring and filter using adisposable filter unit.

Weigh 19.95 g of PEG 400 into a separate jar and place the jar in awater bath at 40° C.±5° C. to warm up. Increase the stirring rate untila vortex forms in the middle of the liquid. Weigh in 50 mg of Compound1, close the jar, and stir the formulation at 40° C.±5° C. until all ofCompound 1 dissolves completely (about 1 hour). Remove the formulationfrom the water bath and stir for at least 1 hour to equilibrate at roomtemperature. Add the vitamin E TPGS solution from the first jar and mixthe formulation by hand until a homogeneous solution forms.

Preparation of a 40 g Formulation Comprising 100 mg of Compound 1

A 40 g aqueous formulation is prepared with the following components andamounts:

Amount Component (g) Compound 1 0.10 PEG 400 19.90 Vitamin E TPGS 4.48Water 15.52

A 22.4% by weight vitamin E TPGS solution is prepared as describedabove.

Weigh 19.90 g of PEG 400 into a separate jar and place the jar in awater bath at 40° C.±5° C. to warm up. Increase the stirring rate untila vortex forms in the middle of the liquid. Weigh in 100 mg of Compound1, close the jar, and stir the formulation at 40° C.±5° C. until all ofCompound 1 dissolves completely (about 1 hour). Remove the formulationfrom the water bath and stir for at least 1 hour to equilibrate at roomtemperature. Add the vitamin E TPGS solution from the first jar and mixthe formulation by hand until a homogeneous solution forms.

Preparation of a 40 g Formulation Comprising 200 mg of Compound 1

A 40 g aqueous formulation is prepared with the following components andamounts:

Amount Component (g) Compound 1 0.20 PEG 400 19.80 Vitamin E TPGS 4.48Water 15.52

A 22.4% by weight vitamin E TPGS solution is prepared as describedabove.

Weigh 19.80 g of PEG 400 into a separate jar and place the jar in awater bath at 40° C.±5° C. to warm up. Increase the stirring rate untila vortex forms in the middle of the liquid. Weigh in 200 mg of Compound1, close the jar, and stir the formulation at 40° C.±5° C. until all ofCompound 1 dissolves completely (about 1 hour). Remove the formulationfrom the water bath and stir for at least 1 hour to equilibrate at roomtemperature. Add the vitamin E TPGS solution from the first jar and mixthe formulation by hand until a homogeneous solution forms.

Preparation of a 40 g Formulation Comprising 300 mg of Compound 1

A 40 g aqueous formulation is prepared with the following components andamounts:

Amount Component (g) Compound 1 0.30 PEG 400 19.70 Vitamin E TPGS 4.48Water 15.52

A 22.4% by weight vitamin E TPGS solution is prepared as describedabove.

Weigh 19.70 g of PEG 400 into a separate jar and place the jar in awater bath at 40° C.±5° C. to warm up. Increase the stirring rate untila vortex forms in the middle of the liquid. Weigh in 300 mg of Compound1, close the jar, and stir the formulation at 40° C.±5° C. until all ofCompound 1 dissolves completely (about 1 hour). Remove the formulationfrom the water bath and stir for at least 1 hour to equilibrate at roomtemperature. Add the vitamin E TPGS solution from the first jar and mixthe formulation by hand until a homogeneous solution forms.

Assays

Assays for Detecting and Measuring ΔF508-CFTR Correction Properties ofCompounds

Membrane Potential Optical Methods for Assaying ΔF508-CFTR ModulationProperties of Compounds

The optical membrane potential assay utilized voltage-sensitive FRETsensors described by Gonzalez and Tsien (See Gonzalez, J. E. and R. Y.Tsien (1995) “Voltage sensing by fluorescence resonance energy transferin single cells” Biophys J 69(4): 1272-80, and Gonzalez, J. E. and R. Y.Tsien (1997) “Improved indicators of cell membrane potential that usefluorescence resonance energy transfer” Chem Biol 4(4): 269-77) incombination with instrumentation for measuring fluorescence changes suchas the Voltage/Ion Probe Reader (VIPR) (See, Gonzalez, J. E., K. Oades,et al. (1999) “Cell-based assays and instrumentation for screeningion-channel targets” Drug Discov Today 4(9): 431-439).

These voltage sensitive assays are based on the change in fluorescenceresonant energy transfer (FRET) between the membrane-soluble,voltage-sensitive dye, DiSBAC₂(3), and a fluorescent phospholipid,CC2-DMPE, which is attached to the outer leaflet of the plasma membraneand acts as a FRET donor. Changes in membrane potential (V_(m)) causethe negatively charged DiSBAC₂(3) to redistribute across the plasmamembrane and the amount of energy transfer from CC2-DMPE changesaccordingly. The changes in fluorescence emission were monitored usingVIPR™ II, which is an integrated liquid handler and fluorescent detectordesigned to conduct cell-based screens in 96- or 384-well microtiterplates.

1. Identification of Correction Compounds

To identify small molecules that correct the trafficking defectassociated with ΔF508-CFTR; a single-addition HTS assay format wasdeveloped. The cells were incubated in serum-free medium for 16 hrs at37° C. in the presence or absence (negative control) of test compound.As a positive control, cells plated in 384-well plates were incubatedfor 16 hrs at 27° C. to “temperature-correct” ΔF508-CFTR. The cells weresubsequently rinsed 3× with Krebs Ringers solution and loaded with thevoltage-sensitive dyes. To activate ΔF508-CFTR, 10 μM forskolin and theCFTR potentiator, genistein (20 μM), were added along with Cl⁻-freemedium to each well. The addition of Cl⁻-free medium promoted Cl⁻ effluxin response to ΔF508-CFTR activation and the resulting membranedepolarization was optically monitored using the FRET-basedvoltage-sensor dyes.

2. Identification of Potentiator Compounds

To identify potentiators of ΔF508-CFTR, a double-addition HTS assayformat was developed. During the first addition, a Cl⁻-free medium withor without test compound was added to each well. After 22 sec, a secondaddition of Cl⁻-free medium containing 2-10 μM forskolin was added toactivate ΔF508-CFTR. The extracellular Cl⁻ concentration following bothadditions was 28 mM, which promoted Cl⁻ efflux in response to ΔF508-CFTRactivation and the resulting membrane depolarization was opticallymonitored using the FRET-based voltage-sensor dyes.

3. Solutions

-   -   Bath Solution #1: (in mM) NaCl 160, KCl 4.5, CaCl₂ 2, MgCl₂ 1,        HEPES10, pH 7.4 with NaOH.    -   Chloride-free bath solution: Chloride salts in Bath Solution #1        are substituted with gluconate salts.    -   CC2-DMPE: Prepared as a 10 mM stock solution in DMSO and stored        at −20° C.    -   DiSBAC₂(3): Prepared as a 10 mM stock in DMSO and stored at −20°        C.

4. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used foroptical measurements of membrane potential. The cells are maintained at37° C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAA, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For all opticalassays, the cells were seeded at 30,000/well in 384-well matrigel-coatedplates and cultured for 2 hrs at 37° C. before culturing at 27° C. for24 hrs for the potentiator assay. For the correction assays, the cellsare cultured at 27° C. or 37° C. with and without compounds for 16-24hours.

Electrophysiological Assays for Assaying ΔF508-CFTR ModulationProperties of Compounds

1. Ussing Chamber Assay

Using chamber experiments were performed on polarized epithelial cellsexpressing ΔF508-CFTR to further characterize the ΔF508-CFTR modulatorsidentified in the optical assays. FRT^(ΔF508-CFTR) epithelial cellsgrown on Costar Snapwell cell culture inserts were mounted in an Ussingchamber (Physiologic Instruments, Inc., San Diego, Calif.), and themonolayers were continuously short-circuited using a Voltage-clampSystem (Department of Bioengineering, University of Iowa, Iowa, and,Physiologic Instruments, Inc., San Diego, Calif.). Transepithelialresistance was measured by applying a 2-mV pulse. Under theseconditions, the FRT epithelia demonstrated resistances of 4 (KΩ/cm² ormore. The solutions were maintained at 27° C. and bubbled with air. Theelectrode offset potential and fluid resistance were corrected using acell-free insert. Under these conditions, the current reflects the flowof Cl⁻ through ΔF508-CFTR expressed in the apical membrane. The I_(SC)was digitally acquired using an MP100A-CE interface and AcqKnowledgesoftware (v3.2.6; BIOPAC Systems, Santa Barbara, Calif.).

2. Identification of Correction Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringer was usedon the basolateral membrane, whereas apical NaCl was replaced byequimolar sodium gluconate (titrated to pH 7.4 with NaOH) to give alarge Cl⁻ concentration gradient across the epithelium. All experimentswere performed with intact monolayers. To fully activate ΔF508-CFTR,forskolin (10 μM) and the PDE inhibitor, IBMX (100 μM), were appliedfollowed by the addition of the CFTR potentiator, genistein (50 μM).

As observed in other cell types, incubation at low temperatures of FRTcells stably expressing ΔF508-CFTR increases the functional density ofCFTR in the plasma membrane. To determine the activity of correctioncompounds, the cells were incubated with 10 μM of the test compound for24 hours at 37° C. and were subsequently washed 3× prior to recording.The cAMP- and genistein-mediated I_(SC) in compound-treated cells wasnormalized to the 27° C. and 37° C. controls and expressed as percentageactivity. Preincubation of the cells with the correction compoundsignificantly increased the cAMP- and genistein-mediated I_(SC) comparedto the 37° C. controls.

3. Identification of Potentiator Compounds

Typical protocol utilized a basolateral to apical membrane Cl⁻concentration gradient. To set up this gradient, normal ringers was usedon the basolateral membrane and was permeabilized with nystatin (360μg/ml), whereas apical NaCl was replaced by equimolar sodium gluconate(titrated to pH 7.4 with NaOH) to give a large Cl⁻ concentrationgradient across the epithelium. All experiments were performed 30 minafter nystatin permeabilization. Forskolin (10 μM) and all testcompounds were added to both sides of the cell culture inserts. Theefficacy of the putative ΔF508-CFTR potentiators was compared to that ofthe known potentiator, genistein.

-   -   4. Solutions

Basolateral solution (in mM): NaCl (135), CaCl₂ (1.2), MgCl₂ (1.2),K₂HPO₄ (2.4), KHPO₄ (0.6),N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES) (10), anddextrose (10). The solution was titrated to pH 7.4 with NaOH.

-   -   Apical solution (in mM): Same as basolateral solution with NaCl        replaced with Na Gluconate (135).

5. Cell Culture

Fisher rat epithelial (FRT) cells expressing ΔF508-CFTR(FRT^(ΔF508-CFTR)) were used for Ussing chamber experiments for theputative ΔF508-CFTR modulators identified from our optical assays. Thecells were cultured on Costar Snapwell cell culture inserts and culturedfor five days at 37° C. and 5% CO₂ in Coon's modified Ham's F-12 mediumsupplemented with 5% fetal calf serum, 100 U/ml penicillin, and 100μg/ml streptomycin. Prior to use for characterizing the potentiatoractivity of compounds, the cells were incubated at 27° C. for 16-48 hrsto correct for the ΔF508-CFTR. To determine the activity of correctionscompounds, the cells were incubated at 27° C. or 37° C. with and withoutthe compounds for 24 hours.

6. Whole-Cell Recordings

The macroscopic ΔF508-CFTR current (I_(ΔF508)) in temperature- and testcompound-corrected NIH3T3 cells stably expressing ΔF508-CFTR weremonitored using the perforated-patch, whole-cell recording. Briefly,voltage-clamp recordings of I_(ΔF508) were performed at room temperatureusing an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.,Foster City, Calif.). All recordings were acquired at a samplingfrequency of 10 kHz and low-pass filtered at 1 kHz. Pipettes had aresistance of 5-6 MΩ when filled with the intracellular solution. Underthese recording conditions, the calculated reversal potential for Cl⁻(E_(Cl)) at room temperature was −28 mV. All recordings had a sealresistance >20 GΩ and a series resistance<15 MΩ. Pulse generation, dataacquisition, and analysis were performed using a PC equipped with aDigidata 1320 A/D interface in conjunction with Clampex 8 (AxonInstruments Inc.). The bath contained <250 μl of saline and wascontinuously perifused at a rate of 2 ml/min using a gravity-drivenperfusion system.

7. Identification of Correction Compounds

To determine the activity of correction compounds for increasing thedensity of functional ΔF508-CFTR in the plasma membrane, we used theabove-described perforated-patch-recording techniques to measure thecurrent density following 24-hr treatment with the correction compounds.To fully activate ΔF508-CFTR, 10 μM forskolin and 20 μM genistein wereadded to the cells. Under our recording conditions, the current densityfollowing 24-hr incubation at 27° C. was higher than that observedfollowing 24-hr incubation at 37° C. These results are consistent withthe known effects of low-temperature incubation on the density ofΔF508-CFTR in the plasma membrane. To determine the effects ofcorrection compounds on CFTR current density, the cells were incubatedwith 10 μM of the test compound for 24 hours at 37° C. and the currentdensity was compared to the 27° C. and 37° C. controls (% activity).Prior to recording, the cells were washed 3× with extracellularrecording medium to remove any remaining test compound. Preincubationwith 10 μM of correction compounds significantly increased the cAMP- andgenistein-dependent current compared to the 37° C. controls.

8. Identification of Potentiator Compounds

The ability of ΔF508-CFTR potentiators to increase the macroscopicΔF508-CFTR Cl⁻ current (I_(ΔF508)) in NIH3T3 cells stably expressingΔF508-CFTR was also investigated using perforated-patch-recordingtechniques. The potentiators identified from the optical assays evoked adose-dependent increase in I_(ΔF508) with similar potency and efficacyobserved in the optical assays. In all cells examined, the reversalpotential before and during potentiator application was around −30 mV,which is the calculated E_(Cl) (−28 mV).

9. Solutions

-   -   Intracellular solution (in mM): Cs-aspartate (90), CsCl (50),        MgCl₂ (1), HEPES (10), and 240 μg/ml amphotericin-B (pH adjusted        to 7.35 with CsOH).    -   Extracellular solution (in mM): N-methyl-D-glucamine (NMDG)-Cl        (150), MgCl₂ (2), CaCl₂ (2), HEPES (10) (pH adjusted to 7.35        with HCl).

10. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forwhole-cell recordings. The cells are maintained at 37° C. in 5% CO₂ and90% humidity in Dulbecco's modified Eagle's medium supplemented with 2mM glutamine, 10% fetal bovine serum, 1×NEAR, β-ME, 1× pen/strep, and 25mM HEPES in 175 cm² culture flasks. For whole-cell recordings,2,500-5,000 cells were seeded on poly-L-lysine-coated glass coverslipsand cultured for 24-48 hrs at 27° C. before use to test the activity ofpotentiators; and incubated with or without the correction compound at37° C. for measuring the activity of correctors.

11. Single-Channel Recordings

The single-channel activities of temperature-corrected ΔF508-CFTR stablyexpressed in NIH3T3 cells and activities of potentiator compounds wereobserved using excised inside-out membrane patch. Briefly, voltage-clamprecordings of single-channel activity were performed at room temperaturewith an Axopatch 200B patch-clamp amplifier (Axon Instruments Inc.). Allrecordings were acquired at a sampling frequency of 10 kHz and low-passfiltered at 400 Hz. Patch pipettes were fabricated from Corning KovarSealing #7052 glass (World Precision Instruments, Inc., Sarasota, Fla.)and had a resistance of 5-8 MΩ when filled with the extracellularsolution. The ΔF508-CFTR was activated after excision, by adding 1 mMMg-ATP, and 75 nM of the cAMP-dependent protein kinase, catalyticsubunit (PKA; Promega Corp. Madison, Wis.). After channel activitystabilized, the patch was perifused using a gravity-drivenmicroperfusion system. The inflow was placed adjacent to the patch,resulting in complete solution exchange within 1-2 sec. To maintainΔF508-CFTR activity during the rapid perifusion, the nonspecificphosphatase inhibitor F (10 mM NaF) was added to the bath solution.Under these recording conditions, channel activity remained constantthroughout the duration of the patch recording (up to 60 min). Currentsproduced by positive charge moving from the intra- to extracellularsolutions (anions moving in the opposite direction) are shown aspositive currents. The pipette potential (V_(P)) was maintained at 80mV.

Channel activity was analyzed from membrane patches containing 2 activechannels. The maximum number of simultaneous openings determined thenumber of active channels during the course of an experiment. Todetermine the single-channel current amplitude, the data recorded from120 sec of ΔF508-CFTR activity was filtered “off-line” at 100 Hz andthen used to construct all-point amplitude histograms that were fittedwith multigaussian functions using Bio-Patch Analysis software(Bio-Logic Comp. France). The total microscopic current and openprobability (P_(o)) were determined from 120 sec of channel activity.The P_(o) was determined using the Bio-Patch software or from therelationship P_(o)=I/i(N), where I=mean current, i=single-channelcurrent amplitude, and N=number of active channels in patch.

12. Solutions

-   -   Extracellular solution (in mM): NMDG (150), aspartic acid (150),        CaCl₂ (5), MgCl₂ (2), and HEPES (10) (pH adjusted to 7.35 with        Tris base).    -   Intracellular solution (in mM): NMDG-Cl (150), MgCl₂ (2), EGTA        (5), TES (10), and Tris base (14) (pH adjusted to 7.35 with        HCl).

13. Cell Culture

NIH3T3 mouse fibroblasts stably expressing ΔF508-CFTR are used forexcised-membrane patch-clamp recordings. The cells are maintained at 37°C. in 5% CO₂ and 90% humidity in Dulbecco's modified Eagle's mediumsupplemented with 2 mM glutamine, 10% fetal bovine serum, 1×NEAR, β-ME,1× pen/strep, and 25 mM HEPES in 175 cm² culture flasks. For singlechannel recordings, 2,500-5,000 cells were seeded onpoly-L-lysine-coated glass coverslips and cultured for 24-48 hrs at 27°C. before use.

Using the procedures described above, the activity, i.e., IC50s, ofCompound 1 has been measured and is shown in Table 11.

TABLE 11 IC50 Bins: +++ <= 2.0 < ++ <= 5.0 < + PercentActivity Bins: +<= 25.0 < ++ <= 100.0 < +++ Cmpd. No. Binned IC50 Binned MaxEfficacy 1+++ +++

1. A formulation comprising(R)-1-(2,2-difluorobenzo[d][1,3]dioxol-5-yl)-N-(1-(2,3-dihydroxypropyl)-6-fluoro-2-(1-hydroxy-2-methylpropan-2-yl)-1H-indol-5-yl)cyclopropanecarboxamide(Compound 1), water, and a polyethylene glycol (PEG).
 2. The formulationof claim 1, wherein the polyethylene glycol is selected from PEG 300,PEG 400, PEG 500, PEG 600, PEG 700, PEG 800, PEG 900, or PEG
 1000. 3.The formulation of claim 1, wherein the polyethylene glycol is PEG 400.4. The formulation of claim 1, wherein the concentration of Compound 1is from about 0.05 to about 3% by weight.
 5. The formulation of claim 1,wherein the concentration of Compound 1 is from about 0.1 to about 2% byweight.
 6. The formulation of claim 1, wherein the concentration ofCompound 1 is about 0.12, 0.25, or 0.50% by weight.
 7. The formulationof claim 1, wherein the concentration of polyethylene glycol is fromabout 40 to about 60% by weight.
 8. The formulation of claim 1, whereinthe concentration of polyethylene glycol is from about 45 to about 55%by weight.
 9. The formulation of claim 1, wherein the concentration ofpolyethylene glycol is about 50% by weight.
 10. The formulation of claim1, wherein the concentration of Compound 1 is from about 0.05 to about3% by weight; and the concentration of polyethylene glycol is from about40 to about 60% by weight.
 11. The formulation of claim 1, wherein theconcentration of Compound 1 is from about 0.1 to about 2% by weight; andthe concentration of polyethylene glycol is from about 45 to about 55%by weight.
 12. The formulation of claim 1, wherein the concentration ofCompound 1 is about 0.12, 0.25, or 0.50% by weight; and theconcentration of polyethylene glycol is about 50% by weight.
 13. Theformulation of claim 1, wherein the concentration of Compound 1 is about0.12, 0.25, or 0.50% by weight; and the polyethylene glycol is PEG 400at about 50% by weight.
 14. The formulation of claim 1 furthercomprising a surfactant.
 15. The formulation of claim 14, wherein thesurfactant is an anionic, cationic, or nonionic surfactant.
 16. Theformulation of claim 14, wherein the surfactant is a nonionic surfactantselected from the group consisting of vitamin E d-α-tocopheryl PEG 1000succinate (vitamin E TPGS), polysorbate 20, polysorbate 40, polysorbate60, polysorbate 65, polysorbate 80, alkyl poly(ethylene oxide),poloxamine, alkyl polyglucosides, octyl glucoside, decyl maltoside,fatty alcohol, cetyl alcohol, oleyl alcohol, cocamide MEA, cocamide DEA,Solutol HS 15, and cocamide TEA.
 17. The formulation of claim 14,wherein the surfactant is vitamin E TPGS.
 18. The formulation of claim14, wherein the concentration of surfactant is from about 5 to about 15%by weight.
 19. The formulation of claim 14, wherein the concentration ofsurfactant is from about 10 to about 12% by weight.
 20. The formulationof claim 14, wherein the concentration of surfactant is about 11% byweight.
 21. The formulation of claim 14, wherein the surfactant isvitamin E TPGS at about 11% by weight.
 22. The formulation of claim 14,wherein the concentration of Compound 1 is from about 0.05 to about 3%by weight, the concentration of polyethylene glycol is from about 40 toabout 60% by weight, and the concentration of surfactant is from about 5to about 15% by weight.
 23. The formulation of claim 22, wherein theconcentration of Compound 1 is from about 0.05 to about 3% by weight,the polyethylene glycol is PEG 400 at about 40 to about 60% by weight,and the surfactant is vitamin E TPGS at about 5 to 15% by weight. 24.The formulation of claim 1, wherein the formulation is about 30 to 50 g.25. The formulation of claim 1, wherein the formulation is about 35 to45 g.
 26. The formulation of claim 1, wherein the formulation is about40 g.
 27. The formulation of claim 14, wherein the formulation comprisesthe following: Amount Component (g) Compound 1 0.05 PEG 400 19.95Vitamin E TPGS 4.48 Water 15.52


28. The formulation of claim 14, wherein the formulation comprises thefollowing: Amount Component (g) Compound 1 0.10 PEG 400 19.90 Vitamin ETPGS 4.48 Water 15.52


29. The formulation of claims 14, wherein the formulation comprises thefollowing: Amount Component (g) Compound 1 0.20 PEG 400 19.80 Vitamin ETPGS 4.48 Water 15.52


30. The formulation of claim 14, wherein the formulation comprises thefollowing: Amount Component (g) Compound 1 0.30 PEG 400 19.70 Vitamin ETPGS 4.48 Water 15.52


31. The formulation of claim 14, wherein the formulation comprises thefollowing: Amount Component (% by weight) Compound 1 0.10-0.80 PEG 40048-51 Vitamin E TPGS 10-12 Water 37-40


32. The formulation of claim 14, wherein the formulation comprises thefollowing: Amount Component (% by weight) Compound 1 0.20-0.60 PEG 40049-50 Vitamin E TPGS 10-12 Water 37-40


33. The formulation of claim 14, wherein the formulation comprises thefollowing: Amount Component (% by weight) Compound 1 0.25 PEG 400 49.75Vitamin E TPGS 11.20 Water 38.80


34. The formulation of claim 14, wherein the formulation comprises thefollowing: Amount Component (% by weight) Compound 1 0.50 PEG 400 49.50Vitamin E TPGS 11.20 Water 38.80


35. The formulation of claim 1, further comprising a taste masker. 36.The formulation of claim 1, further comprising an additional therapeuticagent.
 37. The formulation of claim 36, wherein the additionaltherapeutic agent is selected from a mucolytic agent, bronchodialator,an anti-biotic, an anti-infective agent, an anti-inflammatory agent, aCFTR modulator other than Compound 1, or a nutritional agent.
 38. Amethod of treating a CFTR mediated disease selected from cysticfibrosis, COPD, emphysema, dry-eye disease or osteoporosis in a subjectcomprising administering to the subject an effective amount of theformulation of claim
 1. 39. The method of claim 38, wherein the CFTRmediated disease is cystic fibrosis.
 40. The method of claim 38, whereinthe method comprises administering an additional therapeutic agent. 41.The method of claim 40, wherein the additional therapeutic agent isselected from a mucolytic agent, bronchodialator, an anti-biotic, ananti-infective agent, an anti-inflammatory agent, a CFTR modulator otherthan Compound 1, or a nutritional agent.
 42. A pharmaceutical pack orkit comprising the formulation of claim 1 and instructions for usethereof.